PROJECT SUMMARY/ABSTRACT ? PROJECT 2 The exercise pressor reflex, which arises from contracting skeletal muscles, is one of the two neural mechanisms that evoke the sympathetic and cardiovascular adjustments to exercise. The abnormalities in this reflex affect blood flow delivery and oxygen supply to exercising muscles in cardiovascular diseases. Peripheral arterial disease (PAD) is a manifestation of common and lethal atherosclerotic vascular disorders. The major goal of this project is to better understand the role of pro-inflammatory cytokines (PICs) in controlling excitability of the thin fiber (group III & IV) muscle afferent neurons that evoke the exercise pressor reflex in PAD. With respect to thin fiber muscle afferents, we propose to combine the power of in vitro whole-cell patch- clamp techniques, which will determine mechanisms of afferent excitability, with the insights provided by in vivo physiology, which will determine how these mechanisms translate into increased excitability. Particular attention will be paid to afferents that have TTX sensitive and resistant Na+ channels. For in vitro experiments, muscle afferents will be identified by labeling dorsal root ganglion (DRG) neurons with the retrograde fluorescent tracer DiI that has been injected into the triceps surae muscles of control rats and rats with chronic occlusion of the femoral artery. Moreover, the isolated DRG cells will be identified by their transient expression of green fluorescent protein, the expression of which is driven by the proximal neuron specific promoter region of the TTX sensitive Na+ channel (NaV1.7) and TTX-resistant Na+ channel (NaV1.8). For the in vivo experiments, sympathetic responsiveness evoked by muscle contraction will be examined in control rats and rats with femoral artery occlusion. In both in vitro and in vivo experiments, particular attention will be paid to the effects of PICs, namely IL-6 and TNF-?, on the membrane and discharge properties of the afferent neurons. Also, the interplay between cytokine receptors and channels that control excitability of the thin fiber muscle afferents will be examined. Additionally, intracellular signaling pathways that are engaged during excitation by PICs will be examined. Our general hypothesis is that the higher levels of PIC receptors are induced in muscle DRG neurons after hindlimb blood flow occlusion in the rat model of PAD. This in turn alters the intracellular transduction pathways and the expression and function of Na+ channels, thereby leading to the augmented exercise pressor reflex. The proposed experiments are anticipated to provide new information regarding the mechanisms by which the excitability of the afferent arm of the exercise pressor reflex is affected by PICs in PAD.